ABSTRACT Tuberculosis (TB) is now the leading infectious disease cause of death worldwide, killing one person every 21 seconds. The growing burden of detected rifampicin-resistant (RR) and multidrug-resistant (MDR)-TB is complicating TB prevention, surveillance and care. MDR-TB is caused by Mycobacterium tuberculosis (M.tb) strains that are resistant to isoniazid (INH) and rifampicin (RIF). Extensively drug resistant (XDR)-TB is a severe form of MDR-TB that involves MDR M.tb strains that are additionally resistant to any fluoroquinolone (FQ) and a second-line injectable drug (kanamycin, amikacin, or capreomycin). M.tb strains causing extreme drug resistant (XXDR)-TB are resistant to all 1st and 2nd line TB drugs. Under programmatic conditions in endemic countries, sputum is collected and cultured every month after drug resistant TB treatment initiation, to further perform the drug susceptibility test to determine if the patient is responding or not to the treatment. In many endemic areas (urban and rural), this process takes between 42-60 days. Here, we propose to use an agar-layer based platform (the 2nd Generation Color Plate test), that can shorten this period to ?14 days. Although our agar-layer test is not a molecular based assay as some of the PCR-based instruments currently being used to diagnose drug susceptible and rifampicin resistant TB, our test can diagnose phenotypic resistance for 11 anti-TB drugs, thus making it unique for this purpose. Based in our publications and preliminary data, our premise is that our 2nd Generation culture based Color Plate test will be able to shorten the lapse time of current culture methods to determine if a drug-resistant TB patient is responding well to anti- TB treatment. This novel test is of particular interest to NIH/NIAID as it may overcome limitations such as cost and infrastructure associated with current molecular diagnostic approaches; and can provide expanded rapid drug susceptibility testing (DST) for 11 anti-TB drugs; including bedaquiline and delamanid, two newly approved anti-TB drugs to treat MDR/XDR-TB cases, with the added problem that bedaquiline and delamanid resistant XXDR-TB cases already have been reported and thus, the urgent need of DST monitoring for these two drugs. We now propose to test this novel test in real life conditions in a high TB, HIV associated TB and MDR-TB country (Mozambique). Partnering with The Manhia Health Research Center (Centro de Investigao em Sade de Manhia, CISM), a well-established research center in Mozambique, we propose: i) To evaluate the diagnostic accuracy of the 2nd Generation Color Plate test in diagnosing drug resistant TB; and ii) Testing the efficacy of the 2nd Generation Color Plate test in monitoring if a patient infected with drug resistant TB is responding to the treatment. We will determine the efficacy and the turnaround of the results of this test, and compare our results with other current diagnostic tests for drug resistant TB testing (GeneXpert MTB/RIF, LPA and BACTECTM MGIT DST).